The long-term goal of the proposed research is to address the biological and methodological issues involved in achieving normal hemostasis for individuals with hemophilia B. Recombinant adenoviral vectors that express the factor IX cDNA when transduced into hepatocytes of hemophilia B dogs result in normal plasma concentrations of the clotting protein and normal hemostasis for several weeks. Gene expression is limited secondary to an immune response directed against the transduced cells. Retroviral- mediated hepatic gene transfer results in permanent expression of factor IX in deficient dogs, but the level of expression is less that 1%. For retroviral-mediated gene transfer, 50- to 100- fold improvement in gene transfer and/or expression will be needed before it will be possible to achieve therapeutic concentrations of factor IX in patients. In this grant application, the studies are proposed to improve retroviral- mediated factor IX gene transfer, and second, to increase the persistence of adenoviral-mediated gene expression from hepatocytes. The specific aims are to: I. Optimize gene expression in factor IX vectors. This will be accomplished by determining the effects of different cis DNA elements on factor IX gene expression in vivo such as: 1) a rearranged albumin promoter; 2) specific introns and 3) the factor IX 3' untranslated region. II. Improve retroviral-mediated hepatic gene delivery for factor IX deficiency. Three approaches will be used: 1) determine whether or not retroviral titer is rate-limiting for hepatocyte transduction in vivo; 2) develop drug selection as a means of expanding the genetically modified hepatocytes in vivo; and, 3) develop a new method of liver regeneration to increase the number of genetically modified hepatocytes in vivo.